Question

7. The DNA (or RNA) sample loading buffer contains Tris, acetate and EDTA and has a pH of 8.0. In addition, the loading buffer also contains 5% glycerol and bromophenol blue. Tris and acetate are buffer to control the solution pH at 8.0, EDTA is a DNase inhibitor which prevents DNA from digesting by DNase. So, what are the purposes of having 5% glycerol and bromophenol blue in the sample loading buffer?

Answer 1

The DNA sample loading buffer contains Glycerol to increase the density of the sample. When DNA samples are loaded onto an Agarose gel, we require the DNA sample to settle at the bottom of the Gel, and not diffuse into the Gel Electrophoresis buffer. Glycerol increases the density of the DNA sample solution, which causes the DNA sample to settle at the bottom of the well and not diffuse into the surrounding buffer, giving a sharp migration pattern, without diffuse lanes/bands.

The purpose of adding Bromophenol Blue is to provide a visual cue about the degree of sample migration during electrophoresis. When performing Agarose Gel Electrophoresis of DNA/RNA samples, DNA is visualized at the end using DNA intercalating dyes like Ethidium Bromide or CyberSafe. However, while the gel is running, DNA cannot be visualized in the same way.

Bromophenol blue is added to the DNA sample as it migrates at approximately the same rate as a 50bp DNA molecule, and the presence of the dye in the sample allows the user to monitor the migration of DNA in the gel and only run the gel for as long as necessary and not longer.